Preparation And Immunocompetence Of Gastric Cancer Specific Nanovaccine

Background Gastric cancer is a common malignancy in manycountries in the world, especially in China. Because of lowefficiency of the conventional treatment, the prognosis ofpatients with gastric cancer is still poor. Cancer vaccine is aneffective method for cancer therapy that was demonstrated by alot of preclinical and clinical research. However, thedevelopment of gastric cancer vaccine was hampered by the lackof specific tumor antigen. MG7-Ag, discovered by our institute,is a new marker of gastric cancer. The epitope of MG7~Ag was foundto locate in a sugar chain, which suggested that the MG7-Ag wasa kind of carbohydrate antigen. The mimicry peptide of MG7-Ag wasobtained previously by screening the phage display librarytechnology in our lab. In vivo experiment showed that the mimicrypeptide could mimic the primary antigen effectively, whichindicated that mimicry peptide of MG7-Ag was a suitable candidatemolecule for the development of gastric cancer vaccine.CpG oligodeoxynucleotides (CpG ODN) is a kind of new high efficient immune adjuvant. Recent research has shown: CpG DNA and/or synthetic CpG oligodeoxynucleotides (CpG ODN) having potent adjuvant activity for a wide range of antigens, It can induce both strong innate and specific immunity and shows promising adjuvant effect for tumor antigen. Besides, CpG ODN can be used alone to activate the innate immunity and trigger a tumor-specific immune response. Of particular interest is their improved activity when closely associated with the antigen.Most of antigen mimitopes are composed of 8-12 peptides, which are degraded easily in vivo and require vector to protect them, so as to enhance the ability of antigen presentation and activate specific immune response. Nanoemulsion is a hypoviscosity, single optically isotropic and thermodynamically stable liquid solution with smaller diameter of 0.1-100nm,, Composed of biological materials, it also has reduced toxicity and is more efficiency. It is potentially becoming a focus point as a delivery formulation for peptides and DNA drug. Nanoemulsion has many advantages as following: (1)It' s thermodynamically stable and can be sterilized by fileration, it also is easilly to be prepared and stored. (2)It can increase the dissolving capacity of different drugs which are difficult to be dissolved in water or in lipid solutions. It is hypoviscosity so there is no pain when be used as injection solution. ?It' s good dispersity make it easy to be absorbed by human body and so improvethe drug' s efficiency. (4)It can protect the drugs which are easilly hydrolysised, and so prolong the function time of the drug. (5)It has target characteristics and can deliver the drug to defferent organs through controlling the diameter of the nanoemulsion. (6)It can schlep ectogenic peptides into cytoplasm through endocytosis and break through the MHC limit through cross-presentation.Another approach to improve the immunogenicity of the peptides vaccine is through combination of multiple epitopes. Multi-epitope vaccines induces specific immune response aganist each epitope in the vaccine. And the potency of repeated-epitope vaccine could be improved because the epitope' s copy is increased and Thl response is enhanced. When several epitopes are combined together, flanking sequence will affect the potency of the vaccine. Therefore, it is crucial to insert spacers between epitopes. Proper spacer can prvent mutual interference between epitopes and unspecific binding with MHC molecules. What more, it can also improve the synergy funcion between epitopes, enhance the binding of epitopes and MHC molecules and partially prevent the hydrolysis of protease to peptides.The purpose of this study is to construct a nanovaccine co-encapsulated with a gastric cancer specific antigen MG7-Ag mimotope peptides and adjuvant CpG ODN 1645 using new nanotechnology as nanoemulsion and evaluate its immunocompetence.Nanovaccine was prepared using magnetic ultrasound methods. BALB/c mice were immunized and the in vivo effectiveness was evaluated using tumor challenge assay. Part I : Study on preparation and characteristic of nanoemulsion. Objective To study the way of preparation of nanoemulsion with proper diameter and stable charateristics. Methods Plot phase diagram was used to study the composing of nanoemulsion and nanoemulsion was prepared using magnetic and ultrasonic technique. Its chacteristic was tested by centrifuging, dye and electron microscope, etc. MTT assay was used to assay its cytotoxicity. Results We successefully prepared the nanoemulsion according the phase diagram. Dye assay showed that it was water in oil (W/0) with average diameter of 20~30nm, high stability and low cytotoxicity. Conclusion The emulsion we prepared fit the standard of nanoemulsion. It was stable and has ideal diameter. Part II: Preparation of gastric cancer specific nanovaccine and study of its immunocompetence Objective To develop a nanovaccine coencapsulated with one MG7~Ag mimotope and CpG adjuvent using new nano-technology and valuate its immune efficacy using animal experiment. Methods Encapsulate synthesized MG7-Ag mimotope peptide and CpG ODN into nanoemulsion using magnetic and ultrasonic technique. HPLC was used to determine encapsulation efficiency. BALB/c mice were immunized intravenously with nanovaccine. Serum titer of MG7 antibody was determined by ELISA assay. MTT, ELISPOT was performed to test the cytotoxicity ofspleen cells. The protective and tumor inhibiting effect of the nanovaccine was evaluated by tumor cell challenge assay. Results MG7 — Ag nanovaccine was successfully constructed with high stability and encapsulation efficiency. It could induce both cellular and humour immune response to MG7-Ag in mice. Tumor cell challenge showed that the tumor masses formed in the mice immunized with coencapsulation nanovaccine became markedly smaller than those formed in the mice of control groups and the tumor inhibition rate of co-encapsulation group is obiviously high than other groups. Conclusion Nanovaccine based on the coencapsulation of one MG7-Ag mimotope and CpG is immunogenic which can induce specific immunity response against tumor in mice. And the vaccine is partially protective. Partlll Preparation of gastric cancer specific nanovaccine and study of its immunocompetence Objective To develop a nanovaccine coencapsulated with multiple MG7-Ag mimotopes and CpG adjuvent using new nano-technology and compare its immune efficacy with one mimotope nanovaccine using animal experiment. Methods Encapsulate synthesized multiple MG7 mimotope peptide and CpG ODN into nanoemulsion using magnetic and ultrasonic technique. Dialyse method was used to sterilize, HPLC to determine encapsulation efficiency. BALB/c mice were immunized with multi-epitope nanovaccine. ELISA assay was used to determine serum titer of MG7 antibody. MTT, ELISPOT was performed to test the cytotoxicity of spleen cells. The protective and tumor...